Microbial extracellular proteases account for a major part, more than one third, of the total worldwide industrial enzyme sales (Cherry and Fidantsef, 2003). Approximately 90% of the commercial proteases are detergent enzymes (Gupta et al., 2002). Other applications include such as food, feed, leather, pharmaceuticals, diagnostics, waste management and silver recovery.
The commercial detergent preparations currently in use comprise alkaline serine proteases originating from Bacillus species (Maurer, 2004). Variants of the Bacillus enzymes with improved catalytic efficiency and/or better stability towards temperature, oxidizing agents and various washing conditions have been developed through site-directed and/or random mutagenesis. Examples of commercial proteases are such as subtilisin Carlsberg (Alcalase®, Novozymes, DK), subtilisin 309 (Savinase®, Novozymes, DK), Subtilisin 147 (Esperase®, Novozymes, DK), Kannase® (Novozymes, DK); Purafect® (Genencor Inc., USA), Purafect® Ox (Genencor Inc., USA), Properase® (Genencor Inc., USA), and the BLAP S and X series (Henkel, Del.).
Alkaline serine protease genes and enzymes (EC 3.4.21) have been characterized also from eukaryotic organisms, including yeast and filamentous fungi. The use of the fungal serine proteases is known from several patent applications. For example, U.S. Pat. No. 3,652,399 and EP 519229 (Takeda Chemical Industries, Ltd., JP) disclose an alkaline protease from the genus Fusarium (teleomorph) or Gibberella (anamorph) particularly from Fusarium sp. S-19-5 (ATCC 20192, IFO 8884), F. oxysporum f. sp. lini (IFO 5880) or G. saubinetti (ATCC 20193, IFO6608), useful in the formulation of detergent and other cleanser compositions. WO1994025583 (NovoNordisk A/S, DK) discloses an active trypsin-like protease enzyme derivable from a Fusarium species, in particular a strain of F. oxysporum (DSM 2672), and the DNA sequence encoding the same. The amino acid sequence of a novel protease deriving from Fusarium sp. BLB (FERM BP-10493) is disclosed in WO 2006101140 (SODX Co. Ltd, Nakamura). Such detergent compositions may further comprise reversible protease inhibitors for stabilizing the enzyme(s) as disclosed in WO 1992003529 and WO 1992005239 (NovoNordisk A/S, DK) or the catalytically active amino acid sequence of a protease may be linked to a sequence comprising a cellulose binding domain as disclosed in WO 1997028243 (NovoNordisk A/S, DK).
The serine proteases may be used in applications alone or in combination with other hydrolyzing enzymes. For example, WO 88/03946 and WO 89/04361 (Novo Industri A/S, DK) disclose an enzymatic detergent additive and a detergent composition comprising a protease and a lipase, wherein the fungal protease is derived from Fusarium, particularly F. oxysporum or F. solani. WO 1997002753 (NovoNordisk A/S, DK) discloses a method for gentle cleaning of soiled process equipment using such a combination of a protease and a lipase. Combination of a cellulase and a protease, particularly a trypsin-like protease from Fusarium sp. DSM 2672 as a detergent additive or composition is disclosed in WO 1992018599 (NovoNordisk A/S, DK).
Trichoderma species have been described to secrete a wide variety of proteases (reviewed in Kredics et al., 2005). However, only few of them have been characterized. Isolation of a serine protease encoding gene, prb1, from the biocontrol strain T. harzianum (isolate later reclassified as T. atroviride) has been disclosed in Geremia et al. (1993). The T. atroviride prb1 gene sequence was used in cloning the prb1 gene from T. hamatum and T. harzianum (Steyaert et al., 2004) and the tvsp1 gene from T. virens (Pozo et al., 2004). The mature T. atroviride PRB1 and T. virens TVSP1 proteins were expected to have pIs at 8.98 and 9.2, respectively, and molecular weights of 29 kDa. They showed homology with several subtilisin-like serine proteases and were assigned to family S8 of serine proteases. The TrichoEST approach (Suarez et al. 2007) revealed four novel serine proteases P5431 (AM294975), P7129 (AM296482), P8048 (AM294978) and P10261 (AM294980) from a biocontrol fungus T. harzianum CECT 2413 belonging to S8A subfamily of proteases. The T. reesei genome project demonstrated presence of several genes encoding different types of proteases (Martinez et al., 2008; genome.jgi-psf.org/Trire2/Trire2.home.html). The homologue to the prb1 gene encodes a protein having ID 121495.
Characterization of the above Trichoderma serine proteases has been suggested to pave the way for identification of candidate biocontrol genes and improved commercial biocontrol agents. Their applications in other biotechnological processes have not been studied. The alkaline serine protease of T. koningii has been suggested to be applicable in detergent industry since crosslinking with glutaraldehyde resulted in an enzyme preparation stable over a wide range of temperature and pH resistant to inhibition by detergents (Manonmani and Joseph, 1993). However, the enzyme differs from the Prb1-type proteases due to its high molecular weight of 85 kDa. Trichoderma species, such as T. reesei QM9414 is known to secrete also a trypsin-like protease of family S1 having molecular weight of 25 kDa and pI of 7.3, and maximum activity at pH 8 and 50° C. (Dienes et al. 2007). EP 1347045 A1 discloses a family S1 serine protease from T. harzianum. Nucleic acid and amino acid sequences of acid protease NSP24 and NSP25 from T. reesei QM6a have been disclosed in WO 2006073839. The recombinantly produced NSP24 has utility, for example, in preparation of food and feed and in detergents.
Also, alkaline proteases from fungal species such as Tritirachium and Conidiobolus have been reported (reviewed in Anwar and Saleemuddin, 1998).
The socioeconomic challenges and governmental regulations have forced detergent industry to take in consideration many environmental aspects including not only the use of more lenient chemicals, which can be used in minor amounts and therefore leave less environmental waste trails, but also the need of energy saving. Detergent enzymes, particularly proteases, are important ingredient in detergent compositions. The need to save energy by decreasing the washing temperatures and the increased use of synthetic fibers which cannot tolerate high temperatures and current lifestyle have changed customer habits and created a demand for new enzymes, which are effective at low temperatures.
Despite the fact that numerous patent publications, reviews and articles have been published, in which serine proteases from various microorganisms, for example, the low temperature alkaline proteases from actinomycete Nocardiopsis dassonvillei (EP 0290567, Novo Nordisk A/S, DK) and fungal Paecilomyces marquandii (EP 0290569, Novo Nordisk A/S, DK) and the trypsin and chymotrypsin-like activities of cold-tolerant Trichoderma isolates (Antal et al., 2000) are disclosed, there is still a great need for alternative serine proteases, which are suitable for and effective in modifying, degrading and removing proteinaceous materials particularly in low or moderate temperature ranges and which are stable in the presence of detergents with highly varying properties.
Detergent industry is making great advances in adapting its new products to customers' habits and needs, the properties of new textile products and new washing machines. In order to fulfill all varying demands of detergent industry and governmental regulations, new serine protease ingredients for detergent compositions should be able to accomplish their tasks in wide pH and temperature ranges and remain stable in variety of conditions, including mechanical and chemical interventions in combination with a variety of different detergents. It is also desirable that the serine protease can be produced in high amounts, which can be cost-effectively down-stream processed, by easy separation from fermentation broth and mycelia.